Molecular Heterogeneity of the Brain Endothelium

Abstract

The blood-brain barrier (BBB) is part of a neurovascular structure located in the brain's micro vessels, that is essential to maintain brain homeostasis, but prevents the brain uptake of most drugs. Because of its importance in neuro-pharmacotherapy, the BBB has been the subject of extensive research since its discovery over 100 years ago. Major advances in understanding the structure and function of the barrier have been made. Drugs are re-designed to cross the BBB. However, despite these efforts, overcoming the BBB efficiently to treat brain diseases safely remains challenging. The majority of BBB research studies focus on the BBB as a homogenous structure throughout the different brain regions. However, this simplification may lead to an inadequate understanding of the BBB function with significant therapeutic consequences. From this perspective, we analyzed the gene and protein expression profiles of the BBB in the micro vessels from the brains of mice that were isolated from two different brain regions, namely the cortex and the hippocampus. The expression profile of the inter-endothelial junctional protein (claudin-5), three ABC transporters (P-glycoprotein, Bcrp and Mrp-1), and three BBB receptors (lrp-1, TRF and GLUT-1) were analyzed. Our gene and protein analysis showed that the brain endothelium in the hippocampus exhibits different expression profiles compared to the brain cortex. Specifically, brain endothelial cells (BECs) of the hippocampus express higher gene levels of abcb1, abcg2, lrp1, and slc2a1 compared to the BECs of the cortex regions with a trend of increase for claudin-5, while BECs of the cortex express higher gene levels of abcc1 and trf compared to the hippocampus. At the protein levels, the P-gp expression was found to be significantly higher in the hippocampus compared to the cortex, while TRF was found to be up-regulated in the cortex. These data suggest that the structure and function of the BBB are not homogeneous, and imply that drugs are not delivered similarly among the different brain regions. Appreciation of the BBB heterogeneity by future research programs is thus critical for efficient drug delivery and the treatment of brain diseases.

Keywords: GLUT-1; P-gp; Trf; bcrp; blood—brain barrier; brain endothelium; claudin-5; cortex; efflux transporters; hippocampus; intercellular junctions; lrp-1; mrp-1.

Figure 1

Markers of the two brain regions: the cortex and the hippocampus. mRNA levels of the brain region markers were measured in the dissected cortex and hippocampus through real-time PCR. Results are the means ± SEM from three lots of cerebral structures from three mice and the PCR performed in duplicate for each single preparation. Statistical comparisons: * p < 0.05.

Figure 2

The purity of the mice brain endothelial micro vessels isolated from the cortex and the hippocampus. mRNA levels of CD3, Glial fibrillary acid, and α-actin were measured through real-time PCR. Results are the means ± SEM from three lots of brain endothelial micro vessels (each lot represents a pool from five mice) and PCR performed in duplicate for each single preparation.

Figure 3

Levels of the inter-endothelial junctional protein (claudin-5) in mouse brain micro vessels isolated from the cortex and the hippocampus.

Figure 4

Levels of the ABC transporter proteins (P-gp, Bcrp, and Mrp1) in mouse brain micro vessels isolated from the cortex and the hippocampus. (A–C) The relative mRNA levels of abcb1a (A), abcg2 (B), and abcc1 (C) were assessed through real-time PCR. GAPDH was used as an internal standard. Data are represented as (2^−ΔCt). The protein expressions of P-gp (A), Bcrp (B), and Mrp-1 (C) were examined using Western blot analysis. Optical densities were analyzed with Image Lab 6.0.1 software (Bio-Rad) and normalized to GAPDH. Results are the means ± SEM from three lots of brain endothelial micro vessels (each lot represents a pool from five mice). Statistical comparisons: * p < 0.05.

Figure 5

Levels of the receptor (Lrp-1, transferrin receptor and GLUT-1) in mouse brain micro vessels isolated from the cortex and the hippocampus. (A–C) The relative mRNA levels of Lrp1 (A), TRF (B), and slc2a1 (C) were assessed through real-time PCR. GAPDH was used as an internal standard. Data are represented as (2^−ΔCt). The protein expressions of Lrp-1 (A), TRF (B), and GLUT-1 (C) were examined by Western blot. Optical densities were analyzed with Image Lab 6.0.1 software (Bio-Rad) and normalized to GAPDH. Results are the means ± SEM from three lots of brain endothelial micro vessels (each lot represents a pool from five mice). Statistical comparisons: * p < 0.05 and ** p < 0.01.